Elucidation of a key position for acyltransfer activity in[i] Candida parapsilosis[/i] lipase/acyltransferase (CpLIP2) and in [i]pseudozyma Antarctica[/i] lipase a (CAL-a) by rational design

• Published in: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
• Main Authors: Jan, Anne Hélène, Subileau, Maeva, Deyrieux, Charlotte, Perrier, Véronique, Dubreucq, Eric
• Other Authors: Ingénierie des Agro-polymères et Technologies Émergentes (UMR IATE), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Metaglyc 2 project (no. 22008910) BASF
• Format: Article in Journal/Newspaper
• Language: English
• Published: HAL CCSD 2016
• Subjects: lipases/acyltransferases; CpLIP2; CAL-A; transesterification; rational design; biocatalysis; [SDV.IDA]Life Sciences [q-bio]/Food engineering; Antarc*; Antarctica
• Online Access: https://hal.archives-ouvertes.fr/hal-01269389; https://hal.archives-ouvertes.fr/hal-01269389/document; https://hal.archives-ouvertes.fr/hal-01269389/file/Jan-BBA-2016-manuscript_1.pdf; https://doi.org/10.1016/j.bbapap.2015.11.006

Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw > 0.9). The discovery of a new member of this group, CduLAc from C. dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Ps. Antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2 M in CAL-A down to 0.3 M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved.